Breast cancer is one of the most common cancers in women, yet even with a stabilizing incidence rate and decreased mortality, more than 180,000 women will be diagnosed and more than 40,000 women will die in the U.S. from the disease this year. A meta-analysis demonstrated an almost 2-fold increased risk for women with at least one first-degree relative diagnosed with breast cancer and there is decreased survival for breast cancer among women with a first-degree relative who also had a poor prognosis. BRCA1 and BRCA2 mutations are strongly associated with familial breast cancer however these mutations are found in only a small percentage of women with breast cancer. Recent genome-wide association studies have identified several polymorphisms associated with breast cancer although risks from these polymorphisms are modest. Our group is testing the hypothesis that certain DNA modifications are associated with breast cancer risk. Such modifications may be determined by both environmental and genetic factors, and would be expected to change over a womans lifetime. Methylation: Our laboratory has established pyrosequencing to quantify methylation at multiple CpG sites in the promoter regions of 4 genes (BRCA1, RASSF1A, RAR&#946;, and GSTP1) shown to have frequent aberrant methylation in breast cancer and, to measure global methylation, in LINE-1 repetitive elements scattered across the genome. We expect to have completed pyrosequencing analysis of all Sister Study samples by summer 2009. Telomeres: Our laboratory has optimized a real-time quantitative PCR technique for estimating relative telomere length. Using this technique the number of telomere priming events, and thus the telomere amplification product, is proportional to telomere length. The method has been validated against traditional terminal restriction fragment (TRF) Southern-based analysis of telomere length, requires much less DNA, and can be efficiently done in epidemiologic studies. We are one of the first groups to employ a modified method that uses a GC clamp on the single copy gene primers in order to temperature-shift the melting point of the product to 89C. This allows us to measure both telomere and the reference single copy gene product in the same tube, thus improving the precision of the estimate and decreasing the amount of DNA required for the assay. We have adapted the method from 96- to 384-well format in order to facilitate the high throughput analysis required for epidemiology studies. Using gamma irradiation and &#947;H2A.X foci formation we have characterized double strand break repair phenotypes for 5 non-synonymous SNPs predicted to be damaging by in silico methods. &#947;H2A.X foci formation proved to be a sensitive indicator of DSBs, doubling above baseline values after only 0.5 Gy of gamma irradiation. The number of foci returned to baseline within 24 hours of a 1.5 Gy dose, but the size and intensity of foci at 24 hours remained significantly elevated suggesting that these features are more persistent markers of exposure. None of the nsSNPs in ATM, BRCA1, LIG4, PNKP, and WRN showed evidence of any effects on damage and repair phenotypes compared to controls. This was surprising to us because all these amino acid changes had been predicted to be damaging by one or more of the in silico functional prediction methods that we used: SIFT, PolyPhen, or SNPs3D (Markunas et al, 2008).